Topical or transdermal drug delivery has several advantages over the conventional oral and intravenous dosage forms. Some advantages of topical or transdermal drug delivery are the prevention of first pass metabolism, minimization of pain, and possible controlled release of drugs. There is a growing interest in the optimization of drug delivery targeted to a physiological site in the skin. Barry, B. W., Novel Mechanisms and Devices to Enable Successful Transdermal Drug Delivery, Eur. J. Pharm. Sci. 2001, 14, 101-114. Several attempts have been made and are still under investigation to develop a topical formulation for micro and macromolecules for the treatment of skin diseases. However, the success in developing a topical delivery formulation will depend on the ability of a drug to permeate the skin in sufficient quantity to achieve its desired therapeutic effects. Most drug candidates alone are not capable of achieving the desired therapeutic effects because they are unable to cross the stratum corneum, and therefore require physical enhancers or special transporters to enter into the skin. Trommer, H. et al., Overcoming the Stratum Corneum: The Modulation of Skin Penetration, Skin Pharmacol. Physiol. 2006, 19, 106-121.
Chemical enhancers are widely used for the topical delivery of active agents because they modulate the penetration of macromolecules across the skin, but most of them have limited success. Barry, B., Breaching the Skin's Barrier to Drugs, Nat. Biotechnol. 2004, 22, 165-167, and Prausnitz, M. R., et al., Transdermal Drug Delivery, Nat. Biotechnol. 2008, 26, 1261-1268. It has been shown that the permeation of melatonin across the skin can be increased significantly using chemical penetration enhancers in solution formulations. Rachakonda V K, et al., Screening of Chemical Penetration Enhancers for Transdermal Drug Delivery Using Electrical Resistance of Skin, Pharm Res. 2008, 25(11), 2697-704, and Sapra B, et al., Percutaneous Permeation Enhancement by Terpenes: Mechanistic View, AAPS J. 2008, 10(1), 120-32. The use of chemical penetration enhancers, such as fatty alcohols and fatty acids, increased the permeation of melatonin across the skin; however, the enhancement of permeation was dependent on the chemical structure of the enhancers.
Techniques such as electroportation, iontophoresis and microneedles have also been used to enhance the skin permeation of active ingredients. Prausnitz, M. R., et al., Transdermal Drug Delivery, Nat. Biotechnol. 2008, 26, 1261-1268, and Kolli, C. S., et al., Characterization of Solid Maltose Microneedles and Their use for Transdermal Delivery, Pharm. Res. 2008, 25(1), 104-13. However, each of these techniques has its respective problems in terms of toxicity and therapeutic feasibility.
Other delivery techniques include the use of nanoparticle delivery systems, which are known to be biocompatible and protect the active ingredient from degradation. Among the various nanoparticle systems, lipid nanoparticles are thought to be promising as drug carrier systems for skin application. Melt-emulsified nanoparticles based on lipids which are solid at room temperature have several advantages over nanoemulsions, nanosuspensions, mixed micelles, polymeric nanoparticles and liposomes. The advantages of these solid lipid nanoparticles (SLN) are that they protect the active ingredients from enzymatic degradation, prevent transepidermal water loss and release the drugs in a controlled manner for prolonged periods and thereby enhance the therapeutic effect. Jenning, V., et al., Vitamin A-Loaded Solid Lipid Nanoparticles for Topical use: Drug Release Properties, J. Control. Release. 2000, 66, 115-126. The stabilization of chemically unstable drugs by incorporation into a lipid matrix and also sustained release is possible due to the solid matrix properties of solid lipid nanoparticles (SLN). Souto, E. B., et al., Development of a Controlled Release Formulation Based on SLN and NLC for Topical Clotrimazole Delivery, Int. J. Pharm. 2004, 278, 71-77, Muller, R. H., et al., Cytotoxicity of Solid Lipid Nanoparticles as a Function of the Lipid Matrix and the Surfactant, Pharm. Res. 1997, 14, 458-462, and Wissing, S. A., et al., Investigations on the Occlusive Properties of Solid Lipid Nanoparticles (SLN), J. Comet. Sci. 2001, 52, 313-323.
SLNs are composed of physiological lipids with low toxicity profile and may potentially find application not only in cosmetic and dermatological preparations but even in parenteral and oral drug formulations. Almeida, A. J., et al., Solid Lipid Nanoparticles as a Drug Delivery System for Peptides and Proteins, Adv. Drug Deliv. Rev. 2007, 59, 478-490 and Muller, R. H., et al., Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC) in Cosmetic and Dermatological Preparations. Adv. Drug Deliv. Rev. 2002, 54, 131-155. Many drugs have been successfully incorporated into SLNs, but their use is restricted due to low drug loading, and drug expulsion from the carrier which leads to a decrease in the chemical stability of the drug molecule during storage.
In order to decrease the degree of organization of the lipid matrix in SLN, and the drug loading, carrier nano lipid crystal nanoparticles (NLCN) were developed and are known as the second generation of lipid nanoparticles. Pardeike, J., et al., Lipid Nanoparticles (SLN, NLC) in Cosmetic and Pharmaceutical Dermal Products, Int. J. Pharm. 366 (1-2) (2009) 170-184. Based on the chemical nature of the lipid molecules, the inner structure of NLCNs differs from that of SLNs, in that NLCNs are composed of mixtures of solid and liquid lipids (oils), whereas SLNs are composed only of solid lipids. Because the solubility of active ingredients in oils is generally much higher than in solid lipids, higher drug loading capacity and minimal expulsion during storage is achieved by NLCNs. Lopes, L. B., et al., Comparative Study of the Skin Penetration of Protein Transduction Domains and a Conjugated Peptide. Pharm. Res. 2005, 22(5), 750-757. While this new generation of NLCNs is believed to enhance delivery, it has been shown that these particles do not cross the stratum corneum barrier. As a result of the occlusive property of the particles, the particles reside in the stratum corneum and release the drug into the epidermis. Wang, J. J., et al., Skin Permeation of Buprenorphine and its Ester Prodrugs from Lipid Nanoparticles: Lipid Emulsion, Nanostructure Lipid Carriers and Solid Lipid Nanoparticles. J. Microencapsul. 2009, 12, 1-14, Fang, J. Y., et al., Lipid Nanoparticles as Vehicles for Topical Psoralen Delivery: Solid Lipid Nanoparticles (SLN) Versus Nanostructured Lipid Carriers (NLC). Eur. J. Pharm. Pharm. Biopharm. 2008, 70 (2), 633-640, and Joshi, M., et al., Nanostructured Lipid Carrier (NLC) Based Gel of Celecoxib. Int. J. Pharm. 2008, 346 (1-2), 124-132. Thus, there is a dire need to develop useful NLCNs to treat skin disorders by permeating the skin and delivering the active substance to a target site that lies in the deep epidermis.
It is well known that it is a difficult task to deliver active substances across the skin due to the barrier function of the skin provided by the highly organized structure of the stratum corneum (SC). The use of several techniques, including cell penetrating peptides (CPP) or cell transduction domains or membrane transduction peptides are emerging as attractive drug delivery tools because of their ability to translocate micro and macromolecules across the cell membrane. Patel, L. N., et al., Cell Penetrating Peptides: Intracellular Pathways and Pharmaceutical Perspectives. Pharm. Res. 2007, 24(11), 1977-92. CPPs have been used to deliver proteins, oligonucleotides, solid lipid nanoparticles, and liposomes into tumor cells. Masterobattista, E., et al., Functional Characterization of an Endosome Disruptive Peptide and its Application in Cystolic Delivery of Immoliposome-Entrapped Proteins. J. Biol. Chem. 2002, 277, 27135-27143, Astriab-Fisher, A., et al., Conjugates of Antisense Oligonucleotides With the Tat and Antenapedia Cell-Penetrating Peptides: Effects on Cellular Uptake, Binding to Target Sequences, and Biologic Actions. Pharm. Res. 2002, 19, 744-754, Rudolph, C., et al., Application of Novel Solid Lipid Nanoparticle (SLN)-Gene Vector Formulations Based on a Dimeric HIV-1-TAT-Peptide In Vitro and In Vivo. Pharm. Res. 2004, 21(9), 1662-1669, and Fretz, M. M. et al., OVCAR-3 Cells Internalize TAT-Peptide Modified Liposomes by Endocytosis. Biochim. Biophys. Acta. 2004, 1665, 48-56. Despite progress in the field, topical and transdermal delivery of peptides and proteins in therapeutics remains difficult.
It has been shown (Lopes, L. B. at 750-757) that CPPs can enhance the skin permeation of cyclosporine and P20 peptides when they are conjugated to the CPP; however, these CPPs are primarily composed of arginine and lysine residues, which confer a positive charge to the CPP. Furthermore, there are several available CPPs that have membrane translocating capability based on their arginine content. Among the cell-penetrating peptides, TAT peptide is known and extensively used for drug delivery. It has been shown that TAT or YARA cell penetrating peptide mediated translocation of P20 peptide across the human skin is very small. Lopes, B. L., at al., Comparative Study of the Skin Penetration Transduction Domains and a Conjugated Peptide, Pharm. Res. 2005, 22, 750-757. However, there is still debate as to whether cell penetrating peptides are capable of translocating the lipid pay load across the skin layers, when the molecule is not conjugated.
None of above discussed references has focused on the delivery of non-conjugated molecules or active substances, or the permeation of CPP surface coated NLCNs into the skin. We have discovered that coating of nanoparticles with CPP enhances the permeation of drug several fold into stratum corneum and epidermis. More particularly, our results show that coating of NLCs with CPP enhances skin permeation of celecoxib by 3-4 fold when compared to non-coated and non-specific CPP coated nanoparticles. Furthermore, we show that the co-localization of FITC labeled CPP and DID encapsulated NLC in the epidermal layers indicates that surface modification of nanoparticles with CPP is an added advantage in delivering a greater amount of nanoparticles or drug into the skin.